CDC42 Gtpase Activation Affects Hela Cell DNA Repair and Proliferation Following UV Radiation-Induced Genotoxic Stress.

Cell division control protein 42 (CDC42) homolog is a small Rho GTPase enzyme that participates in such processes as cell cycle progression, migration, polarity, adhesion, and transcription. Recent studies suggest that CDC42 is a potent tumor suppressor in different tissues and is related to aging processes. Although DNA damage is crucial in aging, a potential role for CDC42 in genotoxic stress remains to be explored. Migration, survival/proliferation and DNA damage/repair experiments were performed to demonstrate CDC42 involvement in the recovery of HeLa cells exposed to ultraviolet radiation-induced stress. Sub-lines of HeLa cells ectopically expressing the constitutively active CDC42-V12 mutant were generated to examine whether different CDC42-GTP backgrounds might reflect different sensitivities to UV radiation. Our results show that CDC42 constitutive activation does not interfere with HeLa cell migration after UV radiation. However, the minor DNA damage exhibited by the CDC42-V12 mutant exposed to UV radiation most likely results in cell cycle arrest at the G2/M checkpoint and reduced proliferation and survival. HeLa cells and Mock clones, which express endogenous wild-type CDC42 and show normal activity, are more resistant to UV radiation. None of these effects are altered by pharmacological CDC42 inhibition. Finally, the phosphorylation status of the DNA damage response proteins γ-H2AX and p-Chk1 was found to be delayed and attenuated, respectively, in CDC42-V12 clones. In conclusion, the sensitivity of HeLa cells to ultraviolet radiation increases with CDC42 over-activation due to inadequate DNA repair signaling, culminating in G2/M cell accumulation, which is translated into reduced cellular proliferation and survival.

Rac1 GTPase-deficient HeLa cells present reduced DNA repair, proliferation, and survival under UV or gamma irradiation.

Rac1 GTPase controls essential cellular functions related to the cytoskeleton, such as motility and adhesion. Rac1 is overexpressed in many tumor cells, including breast cancers, where it is also involved in the proliferation and checkpoint control necessary for the cell's recovery after exposure to ionizing radiation. However, its role in DNA damage and repair remains obscure in other tumor cells and under different genotoxic conditions. Here, we compare HeLa cells with mutants exogenously expressing a dominant-negative Rac1 (HeLa-Rac1-N17) by their responses to DNA damage induced by gamma or UV radiation. In HeLa cells, these treatments led to increased levels of active Rac1 (Rac1-GTP) and of stress fibers, with a diminished ability to migrate compared to untreated cells. However, the reduction of Rac1-GTP in Rac1-N17-deficient clones resulted in much higher levels of polymerized stress fibers accompanied by a strong impairment of cell migration, even after both radiation treatments. With regard to proliferation and genomic stability, dominant-negative Rac1 cells were more sensitive to gamma and UV radiation, exhibiting reduced proliferation and survival consistent with increased DNA damage and delayed or reduced DNA repair observed in this Rac1-deficient clone. The DNA damage response, as indicated by pH2AX and pChk1 levels, was increased in HeLa cells but was not effectively triggered in the Rac1-N17 clone after radiation treatment, which is likely the main cause of DNA damage accumulation. These data suggest that Rac1 GTPase plays an important role in signaling and contributes to the sensitivity of cervical cancer cells under UV or gamma radiation treatments.

RHOAming Through the Nucleotide Excision Repair Pathway as a Mechanism of Cellular Response Against the Effects of UV Radiation.

Typical Rho GTPases include the enzymes RhoA, Rac1, and Cdc42 that act as molecular switches to regulate essential cellular processes in eukaryotic cells such as actomyosin dynamics, cell cycle, adhesion, death and differentiation. Recently, it has been shown that different conditions modulate the activity of these enzymes, but their functions still need to be better understood. Here we examine the interplay between RhoA and the NER (Nucleotide Excision Repair) pathway in human cells exposed to UVA, UVB or UVC radiation. The results show high levels and accumulation of UV-induced DNA lesions (strand breaks and cyclobutane pyrimidine dimers, CPDs) in different cells with RhoA loss of function (LoF), either by stable overexpression of negative dominant RhoA (RhoA-N19 mutant), by inhibition with C3 toxin or by transient silencing with siRNA. Cells under RhoA LoF showed reduced levels of γH2AX, p-Chk1 (Ser345) and p-p53 (Ser15) that reflected causally in their accumulation in G1/S phases, in low survival rates and in reduced cell proliferation, also in accordance with the energy of applied UV light. Even NER-deficient cells (XPA, XPC) or DNA translesion synthesis (TLS)-deficient cells (XPV) showed substantial hypersensitivity to UV effects when previously submitted to RhoA LoF. In contrast, analyses of apoptosis, necrosis, autophagy and senescence revealed that all cells displaying normal levels of active RhoA (RhoA-GTP) are more resistant to UV-promoted cell death. This work reaffirms the role of RhoA protein signaling in protecting cells from damage caused by UV radiation and demonstrates relevant communicating mechanisms between actin cytoskeleton and genomic stability.

Modulation of RhoA GTPase Activity Sensitizes Human Cervix Carcinoma Cells to γ-Radiation by Attenuating DNA Repair Pathways.

Radiotherapy with γ-radiation is widely used in cancer treatment to induce DNA damage reducing cell proliferation and to kill tumor cells. Although RhoA GTPase overexpression/hyperactivation is observed in many malignancies, the effect of RhoA activity modulation on cancer radiosensitivity has not been previously investigated. Here, we generated stable HeLa cell clones expressing either the dominant negative RhoA-N19 or the constitutively active RhoA-V14 and compared the responses of these cell lines with those of parental HeLa cells, after treatment with low doses of γ-radiation. HeLa-RhoA-N19 and HeLa-RhoA-V14 clones displayed reduced proliferation and survival compared to parental cells after radiation and became arrested at cell cycle stages correlated with increased cellular senescence and apoptosis. Also, Chk1/Chk2 and histone H2A phosphorylation data, as well as comet assays, suggest that the levels of DNA damage and DNA repair activation and efficiency in HeLa cell lines are correlated with active RhoA. In agreement with these results, RhoA inhibition by C3 toxin expression drastically affected homologous recombination (HR) and nonhomologous end joining (NHEJ). These data suggest that modulation of RhoA GTPase activity impairs DNA damage repair, increasing HeLa cell radiosensitivity.

The NtrY-NtrX two-component system is involved in controlling nitrate assimilation in Herbaspirillum seropedicae strain SmR1.

Herbaspirillum seropedicae is a diazotrophic ß-Proteobacterium found endophytically associated with gramineae (Poaceae or graminaceous plants) such as rice, sorghum and sugar cane. In this work we show that nitrate-dependent growth in this organism is regulated by the master nitrogen regulatory two-component system NtrB-NtrC, and by NtrY-NtrX, which functions to specifically regulate nitrate metabolism. NtrY is a histidine kinase sensor protein predicted to be associated with the membrane and NtrX is the response regulator partner. The ntrYntrX genes are widely distributed in Proteobacteria. In α-Proteobacteria they are frequently located downstream from ntrBC, whereas in ß-Proteobacteria these genes are located downstream from genes encoding an RNA methyltransferase and a proline-rich protein with unknown function. The NtrX protein of α-Proteobacteria has an AAA+ domain, absent in those from ß-Proteobacteria. An ntrY mutant of H. seropedicae showed the wild-type nitrogen fixation phenotype, but the nitrate-dependent growth was abolished. Gene fusion assays indicated that NtrY is involved in the expression of genes coding for the assimilatory nitrate reductase as well as the nitrate-responsive two-component system NarX-NarL (narK and narX promoters, respectively). The purified NtrX protein was capable of binding the narK and narX promoters, and the binding site at the narX promoter for the NtrX protein was determined by DNA footprinting. In silico analyses revealed similar sequences in other promoter regions of H. seropedicae that are related to nitrate assimilation, supporting the role of the NtrY-NtrX system in regulating nitrate metabolism in H. seropedicae.